PROJECT SUMMARY/ABSTRACT Mast cells are hematopoietic progenitor-derived, granule-containing immune cells that are widely distributed in tissues that interact with the external environment, such as the skin and mucosal tissues. It is well-known that mast cells are significantly involved in IgE-mediated allergic reactions. Mast cell function can also be influenced by IgE-independent mechanisms that involve cytokines, peptides, toll-like receptor ligands, or components of the complement system. The identification of the mast cell surface protein repertoire (e.g., G- protein coupled receptors, receptor tyrosine kinases, integrins, and others) that transduces these stimuli is essential for a comprehensive understanding of the concerted biological function of mast cells in the complex signaling environments generated during homeostasis and disease. However, the mast cell surface proteome has not been comprehensively characterized as of yet. Our preliminary studies indicate that we can use mass spectrometry analysis to identify novel cell surface proteins with the potential to regulate mast cell function. Based on these preliminary investigations, we hypothesize that A) comprehensive analysis of the expression of cell surface molecules in human mast cells can provide relevant information on the signals that influence mast cell function in homeostatic conditions and upon IgE-dependent activation; and B) newly identified cell surface molecules contribute to the ability of mast cells to exert certain functions, such as cell adhesion, pro-inflammatory cytokine production, and pro-wound healing activity. Accordingly, we propose a research plan aimed at characterizing the mast cell surface proteome and at assessing how newly identified mast cell surface proteins influence mast cell function. In Aim 1, we will define the cell surface proteome of nave and IgE/antigen-stimulated human primary mast cells. Specifically, we will use glycoprotein enrichment approaches in in combination with mass spectrometry to determine the cell surface proteome of peripheral blood mononuclear cell-derived human mast cells (PBCMCs), a commonly used model of human primary mast cells. In Aim 2, we will use a CRISPR/Cas9a approach to assess the extent to which newly found cell surface proteins of interest (Fermt3 and Mfge8) contribute to mast cell function, e.g. cell adhesion, pro-inflammatory cytokine production, and pro-wound healing activity, respectively. Together, these studies have the potential to provide an understanding of the mechanisms by which mast cells respond to stimuli in homeostatic conditions and during disease. We are confident that these studies will lay the groundwork for future projects aimed at exploring the therapeutical potential of newly identified pathways and mediators that may inhibit or promote mast cell function in conditions in which mast cells are expected to play detrimental or beneficial roles, such as allergic disorders and bacterial infections, respectively.